biostudies-literatureUnknownAdli MNHGRI NIH HHSBiological mechanisms are inherently dynamic, requiring precise and rapid gene manipulation for effective characterization. Traditional genetic perturbation tools such as siRNA and CRISPR knockout operate on timescales that render them unsuitable for exploring dynamic processes or studying essential genes, where chronic depletion can lead to cell death. Here, we compared four major inducible degron systems-dTAG, HaloPROTAC, and two auxin-inducible degron (AID) tools-in human pluripotent stem cells. We evaluated basal degradation levels, inducible degradation kinetics, and recovery dynamics for endogenously tagged genes. While the AID 2.0 system is the most efficient for rapid protein degradation, it exhibited higher basal degradation and slower recovery after ligand washout. To address theResearch squarers.3.rs-5348956https://www.ebi.ac.uk/biostudies/studies/S-EPMC11601833biostudies-literatureComparative analysis and directed protein evolution yield an improved degron technology with minimal basal degradation, rapid inducible depletion, and faster recovery of target proteins.PMC11601833UM1 HG012649Bai TTekin YAdli MXingNeyisci OPaylakhi SDuval AfalseComparative analysis and directed protein evolution yield an improved degron technology with minimal basal degradation, rapid inducible depletion, and faster recovery of target proteins.Biological mechanisms are inherently dynamic, requiring precise and rapid gene manipulation for effective characterization. Traditional genetic perturbation tools such as siRNA and CRISPR knockout operate on timescales that render them unsuitable for exploring dynamic processes or studying essential genes, where chronic depletion can lead to cell death. Here, we compared four major inducible degron systems-dTAG, HaloPROTAC, and two auxin-inducible degron (AID) tools-in human pluripotent stem cells. We evaluated basal degradation levels, inducible degradation kinetics, and recovery dynamics for endogenously tagged genes. While the AID 2.0 system is the most efficient for rapid protein degradation, it exhibited higher basal degradation and slower recovery after ligand washout. To address the2024-01-01T00:00:00Z2024 Nov2026-04-07T20:28:44.465Z2025-04-06T22:47:40.294ZS-EPMC116018333960649110.21203/rs.3.rs-5348956/v1